Effects of Salt Stress on the Antimicrobial Drug Resistance and Protein Profile of Staphylococcus aureus

Staphylococcus aureus is the causative agent of a high percentage of nosocomially acquired infections and food-borne illnesses. Antimicrobial resistance of S. aureus, especially methicillin-resistant S. aureus (MRSA), continues to be a concernfor clinicians worldwide. Objectives: The aim of this study was to investigate the effects of salt stress on the antimicrobial drug resistance and protein profile of S. aureus. Materials and Methods: Staphylococcus aureus (ATCC 25823) was grown in trypticase soy broth at 370C. Cells in the exponential growth phase were gradually exposed to sub-lethal salt stress with concentrations ranging from 5% to35% (wt/vol). There after, these cells were harvested and re-suspended in a tube containing 0.5mL of saline. To standardize the number of bacteria, the bacterial suspension was compared to the 0.5 McFarland standard suspension. Antibiotic susceptibility was determined using the disk diffusion method, and the method involved plating of cell suspensions with stressed cells and unstressed cells on Mueller-Hinton agar plates. The pooled proteins from each condition were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Results: Compared to the unstressed cells, the cells exposed to salt showed significant changes in resistance to rifampicin (P=O .032), penicillin (P=o.02) and methicillin (P=0.001). Furthermore, SDS-PAGE analysis of pooled proteins from cells exposed to salt showed changes in the protein profile. Conclusions: We conclude that salt stress is responsible for the changes in protein profileand antimicrobial resistance of S. aureus, especially to methicillin. ©2012, AJUMS. Published by Kowsar M-RCo. All rights reserved. Keywords: Staphylococcus aureus. Salt Stress. Antibiotic Resistance. Electrophoresis. Polyacrylamide Gel.

Latest articles from "Jundishapur Journal of Microbiology":

Isolation, Identification and Antimicrobial Susceptibility Pattern of Tatumella ptyseos Strains Isolated From Powdered Infant Formula Milk Consumed in Neonatal Intensive Care Unit: First Report From Iran(June 1, 2014)

In Vitro Study of Potentially Probiotic lactic Acid Bacteria Strains Isolated From Traditional Dairy Products(June 1, 2014)

Optimization of L-Tryptophan Biosynthesis From L-Serine of Processed Iranian Beet and Cane Molasses and Indole by Induced Escherichia coli ATCC 11303 Cells(June 1, 2014)

Survival Rates of Human Immunodeficiency Virus and Tuberculosis Co-Infected Patients(June 1, 2014)

Characterization and Transferring of Human Rotavirus Double-Layered Particles in MA104 Cells(June 1, 2014)

Prevalence of Quinolone Resistance Among Extended-Spectrum [Beta] -Lactamase Producing Uropathogenic Klebsiella pneumoniae(June 1, 2014)

Evaluation of an Iranian Home Helicobacter pylori Stool Antigen ELISA Kit(June 1, 2014)

Other interesting articles:

Common Phenotypic and Genotypic Antimicrobial Resistance Patterns Found in a Case Study of Multiresistant E. coli From Cohabitant Pets, Humans, and Household Surfaces
Journal of Environmental Health (January 1, 2013)

Comparison of the Candida albicans and biofilm formation amount on natural tooth, porcelain and acrylic resin/Dogal dis, porselen ve akrilik reçine üzerindeki Candida albicans tutulumu ve biyofilm olusumu miktarinin karsilastirilmasi
Dicle Tip Dergisi (March 1, 2012)

Cloning, heterologous expression and characterization of three thioredoxin h isoforms (OsTrx1, OsTrx20 and OsTrx23) from rice
Plant Omics (May 1, 2012)

Molecular epidemiology of Multidrug resistant Extended-Spectrum [Beta]-Lactamase Producing Klebsiella pneumoniae outbreak in a neonatal intensive care unit*
International Journal of Collaborative Research on Internal Medicine & Public Health (July 1, 2010)

The First Case of Microsporum persicolor Infection in Iran
Jundishapur Journal of Microbiology (January 1, 2011)

Bacteriuria and Antimicrobial Susceptibility of Escherichia coli Isolated From Urine of Asymptomatic University Students in Keffi, Nigeria
Jundishapur Journal of Microbiology (January 1, 2011)

Taxonomic Characterization and Potential Biotechnological Applications of Yarrowia lipolytica Isolated From Meat and Meat Products
Jundishapur Journal of Microbiology (January 1, 2011)

Publication: Jundishapur Journal of Microbiology
Author: Ganjian, Haleh
Date published: January 1, 2011

1. Background

Staphylococcus aureus is one of the important human pathogens involved in food-related diseases and a common cause of community-associated infection (1, 2). This organism proliferates in food and releases one or more heat-stable enterotoxins, causing food-borne illnesses (3). S. aureus is the most common cause of infections in hospitalized patients and has been a major concern for well over a century (4). The spectrum of diseases caused by this organism is extremely wide, ranging from superficial infections to deep-seated and systemic infections such as pneumonia, endocarditis, osteomyelitis, and sepsis (5). The treatment of staphylococcal infections has become extremely challenging due to the propensity of the organism to rapidly evolve into antibiotic-resistant strains. Antibiotic resistance is an emerging problem worldwide, and widespread use of antibiotics is likely to be the main reason for the increase in antibiotic resistance (6).

Prevalence of resistant S. aureus, especially methicillinresistant S. aureus (MRSA), is increasing in clinical settings. MRSA is one of the most common causes of nosocomial infections. Methicillin, a semi-synthetic penicillinase-resistant penicillin, was introduced in 1960 for the treatment of penicillinase-producing strains of S. aureus; methicillinresistant strains of S. aureus were identified in 1961 (7). Environmental stresses, including temperature, pH, salts, osmotic pressure, and alkaline and acidic conditions can affect the growth rate and population of bacteria (8). To our knowledge, stress and antimicrobial drug resistance has been studied more extensively in Gram-negative bacteria such as Escherichia coli; however, very little is known about these factors with regard to S. aureus.

2. Objectives

The aim of this study was to investigate the effect of environmental stress on the antibiotic susceptibility and protein profile of S. aureus as a prototypical Gram-positive bacterium.

3. Materials and Methods

S. aureus (ATCC 25823) was obtained from the Iranian Research Organization for Science and Technology and grown in trypticase soy broth (TSB; Merck) at 37°C. S. aureus cells in the exponential growth phase were exposed to sub-lethal salt stress by using concentrations ranging from 5% to 35% (wt/vol). The stress-treated cells were then harvested by centrifugation (3,000 xg for 15 min) and were re-cultured on mannitol salt agar (MSA, Merck). Bacteria were re-suspended in a tube containing 0.5ml of saline. To standardize the number of bacteria, 0.5 McFarland standard was prepared by adding 99.5ml of 1% sulfuric acid to 0.5ml of 1.175% BaCl2 solution. Spectrophotometric analysis of the bacterial suspension by using 0.5 McFarland standard revealed that the suspension contained l.5xl08 bacteria per milliliter (CFU/ mL). Cell suspensions containing stressed and normal bacterial cells were plated on Mueller-Hinton agar (MHA; Merck Company) plates using a sterile swab. Susceptibility toll antibiotics was tested by performing the disk diffusion method by using commercial disks (MAST Diagnostics, Merseyside, UK) according to the Clinical Laboratory Standards Institute guidelines (9).

The antibiotics used and their disk potencies were as follows: erythromycin, I5]ig, penicillin G, 1OU; gentamicin, 10]ig; ciprofloxacin, 5]ig, cefalexin, 30]ig, chloramphenicol, 30]ig; co-trimoxazole, 25]ig, rifampicin, 5]ig, clindamycin, 2]ig, cephalothin, 30]ig, and methicillin (25yg). The plates were incubated at 37°C for 17 to 24 h and were then examined 4times for the development of zones of inhibition in each lawn growth around the disc. The pooled protein from stressed and non-stressed (control) bacterial cells were analyzed using sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE) as described by Laemmli (10). Statistical analyses wereperformed by ANOVA by using Statistical Package for Social Sciences (SPSS, versionll.5). AP value of ^0.05 was considered statistically significant.

4. Results

S. aureus ATCC 25823 cultures were gradually adapted to the 4 salt concentrations (5.0%, 15%, 25%, and 35.0% [wt/vol]), in various periods ranging from 5 to 20 min. Evaluation of antimicrobial drug resistance pattern revealed significant differences in zone sizes between the test and control suspensions under salt stress. Salt-stressed S. aureus suspensions had significantly smaller inhibition zonesin the presence of rifampicin, penicillin, and methicillin.However, the zone of gentamicin was bigger to those observed for un-stressed control suspensions (Table). The tested bacterial strain had the highest resistance to methicillin (P<0.001), significantly higher than that shown to any other antibiotics tested, especially in 35% concentration of salt. The SDS-PAGE analysis of pooled proteins at 4 conditions, (i) 5.0%, (ii) 15%, (iii) 25%, and (iv) 35.0% (wt/vol) of salt, in comparison with the control, is shown in Figure 1. Comparison of the protein profiles between control and those treated with different salt concentrations showed that salt treatment decreased the intensity of some of the bands (20 kDa, 40 kDa, and 60 kDa) at salt concentrations of 5% to 35%. In contrast, some protein bands (47 kDa, 66 kDa. and >78 kDa) showed increased intensity in the same range of salt concentrations.

5. Discussion

Historically, salt has been used both as an additive and preservative in foods, and abundant information on it can be found in the literature, salt has often been incorporated as an antimicrobial agent in meat, meat products, or brine solutions (11). In the present study, we showed that S. aureus could grow gradually at 4 salt conditions, (i) 5. 0%, (ii) 15%, (iii) 25%, and (iv) 35. 0% (wt/vol). This finding is consistent those of some previous reports (12, 13). Osmotolerance in this organism is therefore an interesting topic for several reasons. The intracellular concentration of potassium (K) is high and does not change much on salt stress since osmoprotectants can enter S. aureus cells through various transport mechanisms that are activated or induced by salt stress. S. aureus can also activate some genes and express proteins in response to salt stress (14-16). In the present study, antibiotic susceptibility in S. aureus ATCC 25823 decreased in the presence of sub-lethal concentrations of salt. Asimilar response has been reported in wild-type strains of S. aureus isolated from commercial food kitchens (8).

Genotypic changes may be responsible for the development of hyper-resistant varieties of Staphylococcus. By point mutations, target sites for antibiotic binding can be inactivated and heterogeneous populationcan be generated with increased spontaneous mutation rates (hypermutable strains) (8, 17, 18). Our results show that salt stress causes significantly greater changes in methicillin resistance than in any other antibiotics tested. The mechanism behind the staphylococcal resistance to methicillin is attributable to the expression of a unique penicillin-binding protein, PBP2a, which has a much lower affinity for beta-lactam antibiotics (19). Analysis of protein profile in this study shows changes in the protein, which was attributable to the expression and inhibition of some important proteins.

Exposure of microorganisms to sub-lethal concentrations of salt can induce the expression of stress proteins with a profile similar to that of stress protein expression induced by heat shock. Many environmental stresses can induce the Mar (multiple antibiotic resistances) operon that is known to regulate the expression of a large number of genes, including the efflux pump (the arcAB efflux pump) (8, 20). This suggests that using high concentrations of salt in food preservation can lead to the development of population or subpopulation of S. aureus with decreased susceptibility to antibiotics. Results of this study demonstrated that sub-lethal salt stress could significantly alter the antibiotic resistance and protein profile of S. aureus. We conclude that the increased use of salt in food processing may contribute to the development and dissemination of antibiotic resistance S. aureus as food- borne pathogens.


None declared.

Financial disclosure

None declared.


This article is a compilation of results of MS thesis (Code, 20230507882015) from Azad University of Lahijan, Guilan, Iran. The tests were conducted at the Laboratory of Microbiology and Immunology of Infectious Diseases, Paramedicine Faculty, Gilan University of Medical Sciences, Langeroud.


1. Fridkin SK, Hageman JC, Morrison M, Sanza LT, Como-Sabetti K, Jernigan JA, et al. Methicillin-resistant Staphylococcus aureus disease in three communities. N Engl] Med. 2005;352(14):1436^4.

2. Normanno G, La Salandra G, Dambrosio A, Quaglia NC, Corrente M Parisi A, et al. Occurrence, characterization and antimicrobial resistance of enterotoxigenic Staphylococcus aureus isolated from meat and dairy products. IntJ Food Mcrob/o/. 2007;115(3)290-6.

3. Balaban N, Rasooly A Staphylococcal enterotoxins. IntJ Food Microbiol. 2000;61(l)a-10.

4. Ekrami A Samarbafzadeh A Alavi M, Kalantar E, Hamzeloi E Prevalence of methicillin resistant Staphylococcus species isolated from burn patients in a bum center, Ahvaz, Iran. Jundishapur J Microbiol. 201l;3(2):84-91.

5. Bertini G, Nicoletti P, Scopetti F, Manoocher P, Dani C, Orefici G. Staphylococcus aureus epidemic in a neonatal nursery: a strategy of infection control. Eur J Ped/atr. 2006;165(8):530-5.

6. Lietzau S, Sturmer T, Erb A Von Baum H, Marre R, Brenner H. Prevalence and determinants of nasal colonization with antibiotic-resistant Staphylococcus aureus among unselected patients attending general practitioners in Germany. Epidemiol Infect 2004;132(4):655-62.

7. Rahbar M, Safadel N. Evaluation of cefoxitin disk diffusion test for routine detection of methicillin-resistant staphylococcus aureus. Iran J Pathol. 2006;1(4)345-8.

8. McMahon MA Xu J, Moore JE, Blair IS, McDowell DA. Environmental stress and antibiotic resistance in food-related pathogens.<4pp/£nviron Microbiol. 2007;73(l)211-7.

9. Sader HS, Ferrara MJ, Relier LB, Schreckenberger PC, Swenson JM, Jones RN. Révaluation of Clinical and Laboratory Standards Institute disk diffusion breakpoints for tetracyclines for testing Enterobacteriaceae./ Clin Microbiol. 2007;45(5)a640.

10. Nikokar I, Kajbaf M, Mak VM, Farajzadeh A Kamali E, Mostafaei A et al. Isolation and purification of Ag 85 complex from Mycobadvrium Bovis (Beg) and assessment of Its cell proliferation response in whole blood. Yakhteh (The Cell). 2004;6:144-150.

11. Hajmeer M, Ceylan E, Marsden JL, Fung DY. Impact of sodium chloride on Escherichia coli 0157:H7 and Staphylococcus aureus analysed using transmission electron microscopy. Food Microbiol. 2006;23(5):446-52.

12. Tsai M, Ohniwa RL, Kato Y, Takeshita SL, Ohta T, Saito S, et al. Staphylococcus aureus requires cardiolipin for survival under conditions of high salinity. BMC Microbiol. 2011;1103.

13. Vilhelmsson 0, Miller KJ. Synthesis of pyruvate dehydrogenase in Staphylococcus aureus is stimulated by osmotic stress.Appl Environ Microbiol. 2002;68(5):2353-8.

14. Amey DR Phillips CJC. The effects of changes in sodium and potassium concentration on growth of mastiogenic bacteria in vitro. Intern J Appi Res Vet Med. 2005;3:242-8.

15. Scybert S, Pechous R Sitthisak S, Nadakavukaren MJ, Wilkinson BJ, Jayaswal RK NaCl-sensitive mutant of Staphylococcus aureus has a Tn917-lacZ insertion in its ars operan. FEMS Microbiol Lett. 2003;222(2)a71-6.

16. Vijaranakul U, Nadakavukaren MJ, Bayles DO, Wilkinson BJ, Jayaswal RK Characterization of an NaCl-sensitive Staphylococcus aureus mutant and rescue of the NaCl-sensitive phenotype by glycine betaine but not by other compatible solutes. Appi Environ Microbiol. 1997;63(5):l889-97.

17. Livermore DM. Bacterial resistance: origins, epidemiology, and impact. Clin Infect Dis. 2003;36(Suppl l):Sll-23.

18. Martinez JL, Baquero F. Mutation frequencies and antibiotic resistance. Antimicrob Agents Chemother. 2000;44(7):l771-7.

19. Japoni A Alborzi A Orafa F, Rasouli M, Farshad S. distribution patterns of methicillin resistance genes(mecA) in staphylococcus aureus isolated from clinical specimens. Iran Biomed /.2004;8(4):1738.

20. Alekshun MN, Levy SB. Alteration of the repressor activity of MarR the negative regulator of the Escherichia coli marRAB locus, by multiple chemicals in vitro.; Bacteriol. 1999;181(15):4669-72.

Author affiliation:

Haleh Ganjian1, Iraj Nikokar2*, Azita Tieshayar2, Ali Mostafaei3, Nour Amirmozafari4, Sara Kiani3

1 Azad University of Lahijan, Guilan, IR Iran

2 Laboratory of Microbiology and Immunology of Infectious Diseases, Paramedicine Faculty, Guilan University of Medical Sciences, Guilan, IR Iran

3 Kermanshah University of Medical Sciences, Kermanshah, IR Iran

4 Tehran University of Medical Sciences, Tehran, IR Iran

Author affiliation:

* Corresponding author: Iraj Nikokar, Laboratory of Microbiology and Immunology of Infectious Diseases, Paramedicine Faculty, Guilan University of Medical Sciences, P.O. Box: 44715-1361, Langeroud, IR Iran. Tel: +981425237070, Fax: +98-1425237171, Email: Nikokariraj@yahoo.com, Nikokariraj@gums.ac.ir

DOI: 105812/kowsan20083645^375

©2012, AJUMS. Published by Kowsar M-PCo. All rights reserved.

The use of this website is subject to the following Terms of Use